Amyotrophic lateral sclerosis (ALS, also motor neuron or Lou Gehrig's disease) and frontotemporal lobar dementia (FTLD) are debilitating neurodegenerative disorders that are now linked to TAR DNA-binding protein-43 (TDP-43). It is estimated that half of FTLD patients have associated TDP-43 pathology, making TDP-43-associated FTLD the largest single subtype. A key advance in the understanding of the pathogenesis of ALS, and especially sporadic ALS (sALS, ~85-90% of patients), was the discovery that TDP-43 is a key component of the ubiquitinated inclusions in these patients and that numerous mutations in TDP-43 are linked to both familial ALS and sALS. TDP-43 is a primarily nuclear protein that binds to nucleic acids and is believed to function in a variety of roles in the cell including nuclear transport and nucleic acid processing. In the initial report linking TDP-43 to FTLD and ALS and in multiple confirmatory reports, it has been shown that both TDP- 43 and ubiquitinated TDP-43 are present in intracellular inclusions in the neurons of patients and that this aggregation is accompanied by a lack of TDP-43 in the nucleus. Effective TDP-43 based therapeutics would have a high level of penetration into these populations; however, the precise roles of TDP-43 and how their modulation would effect disease progression are not currently understood. The development of TDP-43 probe molecules will dramatically enhance our understanding of its biology and may lead to the rapid development of therapeutics either by serving as therapeutically useful molecules themselves or enabling the development of second generation assays for TDP-43 function. We have developed the first ever assay for small molecule probe binding to TDP-43 suitable for high throughput compound library screening that looks at the inhibition of the binding of oligonucleotides and have identified four chemotypes that possess the novel ability to inhibit the binding of TDP-43 to oligonucleotides. We will explore the chemical space around these hits in a timely and cost-effective manner by (1) continuing to purchase and evaluate related small-molecule libraries and (2) conducting probe library medicinal chemistry incorporating computational pharmacophore development using the Schrodinger software suite. Compounds with sufficient potency in our primary assay will be tested for their ability to inhibit the CFTR exon 9 splicing ability of TDP-43, to alter the cellular distribution of TDP-43 and to alter the toxicity and distribution of mutant TDP-43s. We will perform industry standard selectivity and metabolic studies to validate these compounds for cellular and in vivo use. Future development of these probe molecules may find utility as biochemical tools for screening compound libraries or examining TDP-43 levels and trafficking in model systems. Imaging tools that binds to TDP-43 would serve as strong preclinical research tools and may lead to a potential biomarker for disease diagnosis and progression in humans. The successful development of probe molecules via the work in this application has enormous potential to revolutionize our understanding of TDP-43. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD) are debilitating neurological disorders for which there are no effective, disease modifying therapies available, meaning that diagnosis carries with it the certainty of a decline in health leading to death. Not only are these diseases incurable, but their reach and devastation is much greater because of the impact upon caregivers and the health care system as ALS and FTLD both result in the need for constant around the clock care in their late stages. Both diseases are now linked a protein called TAR DNA binding protein 43 (TDP-43), which aggregates in the nerve cells of patients, and we propose to generate novel molecules that alter TDP 43's ability to bind to nucleic acids that will serve as the first small molecules probes of its function.